Investigating the role of CD44 and hyaluronate in embryo-epithelial interaction using an in vitro model.

Implantation failure is an important impediment to increasing success rates in assisted reproductive technologies. Knowledge of the cascade of morphological and molecular events at implantation remains limited. Cell surface CD44 and hyaluronate (HA) have been reported in the uterus, but a role in intercellular interaction at implantation remains to be evaluated. Mouse embryos were co-cultured with human Ishikawa endometrial epithelial monolayers over 2 days. Attachment was tenuous during the first 24 h, after which it became stable, leading to breaching of the monolayer. The effects of enzymatically reducing the density of HA, or introducing a function-blocking antibody to CD44, were monitored during progression from weak to stable embryonic attachment. Hyaluronidase-mediated removal of surface HA from the epithelial cells enhanced the speed of attachment, while a similar treatment of embryos had no effect. The antibody to CD44 caused retardation of initial attachment. These results suggest that CD44-HA binding could be employed by embryos during initial docking, but the persistence of HA in epithelial cells might be detrimental to later stages of implantation by retarding attainment of stable attachment.

Studies of the dynamics of nuclear clustering in human syncytiotrophoblast.

Syncytial nuclear aggregates (SNAs), clusters of nuclei in the syncytiotrophoblast of the human placenta, are increased as gestation advances and in pregnancy pathologies. The origins of increased SNAs are unclear; however, a better appreciation of the mechanism may give insight into placental ageing and factors underpinning dysfunction. We developed three models to investigate whether SNA formation results from a dynamic process of nuclear movement and to generate alternative hypotheses. SNA count and size were measured in placental explants cultured over 16 days and particles released into culture medium were quantified. Primary trophoblasts were cultured for 6 days. Explants and trophoblasts were cultured with and without cytoskeletal inhibitors. An in silico model was developed to examine the effects of modulating nuclear behaviour on clustering. In explants, neither median SNA number (108 SNA/mm(2) villous area) nor size (283 µm(2)) changed over time. Subcellular particles from conditioned culture medium showed a wide range of sizes that overlapped with those of SNAs. Nuclei in primary trophoblasts did not change position relative to other nuclei; apparent movement was associated with positional changes of the syncytial cell membrane. In both models, SNAs and nuclear clusters were stable despite pharmacological disruption of cytoskeletal activity. In silico, increased nuclear movement, adhesiveness and sites of cytotrophoblast fusion were related to nuclear clustering. The prominence of SNAs in pregnancy disorders may not result from an active process involving cytoskeleton-mediated rearrangement of syncytial nuclei. Further insights into the mechanism(s) of SNA formation will aid understanding of their increased presence in pregnancy pathologies.

Endometrial apical glycoproteomic analysis reveals roles for cadherin 6, desmoglein-2 and plexin b2 in epithelial integrity.

Implantation failure is one of the major causes of infertility and remains a major barrier to assisted reproduction success. Initial receptivity to implantation is regulated by the endometrial luminal epithelium under maternal hormonal control. Identification of epithelial cell surface components involved in embryo attachment will have translational applications in early pregnancy failure, infertility and contraception. In this study, vectorial biotinylation has been used to characterize the apical glycoproteome of Ishikawa cells, a polarized cell line that serves as a model of the implantation-receptive human endometrial luminal epithelium. Of 46 surface-associated glycoproteins detected by mass spectrometry, half are newly reported in this cell type; a subgroup of these were chosen for evaluation in tissue, and all were shown to be expressed apically in vivo in the mid-secretory (implantation) phase of the menstrual cycle, thus validating the model. Eleven adhesion molecules were detected, some already known to be involved in implantation, others novel. Cadherin 6, desmoglein 2 and plexin b2 were surprisingly found in the apical as well as the lateral membrane domain; their knock-down compromised epithelial integrity. This method of targeting glycosylated apical surface moieties in a polarized epithelial culture model shows excellent selectivity and identifies candidate cell adhesion molecules that are also present in vivo in secretory phase endometrial epithelium.

Altered glycosylation in peri-implantation phase endometrium in women with stages III and IV endometriosis.

BACKGROUND: Endometriosis is a common cause of infertility and pelvic pain. Lectin histochemistry has shown that glycan expression is a sensitive marker of differentiation in the normal endometrium. Endometrial biopsies were taken during the implantation window from women with subfertility and advanced (stage III and IV) endometriosis to evaluate specific glycans bound by lectins from Dolichos biflorus agglutinin (DBA) and Vicia villosa agglutinin (VVA), which detect related but distinct glycan sequences regulated by progesterone action. METHODS: Endometrial tissue from 12 women with subfertility and advanced endometriosis and 11 healthy controls were taken on days 19-24 of the menstrual cycle and processed into either epoxy resin or paraffin wax. Lectin histochemistry was analysed using light microscopy to quantify the amount of glandular reaction product. RESULTS: There was a significant (P = 0.011) reduction in DBA binding to endometrium from patients with endometriosis compared with controls, which was not seen with VVA (P = 0.135). Three stage IV biopsies and one stage III biopsy completely failed to bind DBA and, of these, three showed moderate glandular binding of VVA. DBA and VVA binding differed significantly (P= 0.0039) in the endometriosis specimens whereas in controls no significant difference was detected (P = 0.812). CONCLUSION: Secretory phase glycosylation in women with advanced endometriosis differs from that in healthy women with a reduction in fucosylated N-acetylgalactosamine sequences bound by DBA. Shorter VVA-binding glycans are not significantly affected. In addition to indicating abnormalities of epithelial differentiation, these findings may be directly relevant to implantation failure, as blastocyst attachment requires a critical interaction with the epithelial glycocalyx.

Early stages of implantation as revealed by an in vitro model.

Our limited understanding of the processes underlying steroid hormonal control of human endometrial receptivity is largely due to the lack of a relevant model system. To overcome scarcity of material, we have developed a model in which mouse embryos attach to human Ishikawa cells, which express functional steroid hormone receptors. Blastocysts flushed from day 4 pregnant superovulated mice were transferred to confluent Ishikawa cell monolayers. After 48 h of co-culture, 85% of the blastocysts had attached loosely, but only 40% attached stably to the epithelial cell surface. In contrast, 95% of the embryos attached stably to tissue culture plastic. Thus, weak attachment of a majority of the embryos was followed by stronger adhesion of a smaller proportion. Seventeen percent of the transferred blastocysts modified the epithelial cell surface with loss of MUC1 at the attachment site, extending variably to adjacent epithelial cells. Initially, stable attachment occurred without disruption to the integrity of the epithelial monolayer, but at later stages after the embryo had spread laterally, displacement of subjacent cells was observed. A modest increase in stable attachment, but no changes to MUC1 clearance, was observed after assisted hatching. After 24 h priming of Ishikawa cells by 17beta-oestradiol (OE(2)) followed by 72-h incubation with medroxyprogesterone acetate and OE(2), stable attachment increased from 40 to 70%. Initial attachment is efficient either in the presence or in the absence of hormone; steroid treatment increased the incidence of stable attachment. Implantation failure is predicted to occur in this model when embryos fail to progress from initial to stable attachment.

Differential expression of angiotensin II type 1 and type 2 receptors at the maternal-fetal interface: potential roles in early placental development.

Angiotensin II (Ang II) is locally generated in the placenta and regulates syncytial transport, vascular contractility and trophoblast invasion. It acts through two receptor subtypes, AGTR1 and AGTR2 (AT1 and AT2), which typically mediate antagonising actions. The objectives of this study are to characterise the cellular distribution of AGTR1 and AGTR2 at the maternal-fetal interface and explore the effects on cytotrophoblast turnover. Low levels of AGTR2 mRNA were detected in first trimester placental homogenates using real-time PCR. Immunohistochemistry using polyclonal antibodies against AGTR1 and AGTR2 detected the receptors in first trimester placenta, decidua basalis and villous tip outgrowths in culture. Serial staining with cytokeratin-7 was used to identify extravillous trophoblasts (EVTs). AGTR1 was found in the syncytiotrophoblast microvillous membrane, in a subpopulation of villous cytotrophoblasts, and in Hofbauer cells. AGTR1 was strongly upregulated in cytotrophoblasts in cell columns and villous tip outgrowths, but was absent in interstitial and endovascular EVTs within the decidua. AGTR2 immunostaining was present in Hofbauer cells and villous cytotrophoblasts, but was absent from syncytiotrophoblast. Faint staining was detected in cell column cytotrophoblasts and villous outgrowths, but not in EVTs within the decidua. Both receptors were detected in placental homogenates by western blotting. Ang II significantly increased proliferation of cytotrophoblasts in both villous explants and villous tip outgrowths, but did not affect apoptosis. Blockade of AGTR1 and AGTR2 together abrogated this effect. This study shows specific expression patterns for AGTR1 and AGTR2 in distinct trophoblast populations at the maternal-fetal interface and suggests that Ang II plays a role in placental development and generation of EVTs.

Reproductive glycogenetics--a critical factor in pregnancy success and species hybridisation.

Hybridisation occurs rarely in nature and experiments using interspecific transfer of embryos generally result in implantation failure. Here we show that appropriate glycosylation of the apposing surfaces of endometrium and trophoblast probably is an important factor and may play a critical role in pregnancy success. Examination of closely related species shows that each has its own specific pattern of glycosylation, or glycotype, at the fetomaternal interface and that interacting surfaces appear to show complementarity, suggesting the existence of a glycocode. Studies on a camel/llama hybrid show that for successful implantation to occur, a hybrid must have a placental glycosylation pattern similar to that of the host species, suggesting that the glycocode and appropriate glycosylation may be critical factors in the establishment and maintenance of pregnancy. This new field of reproductive glycogenetics is not only relevant to the development of new species but may also have important implications in the area of human fertility.

Adhesion molecules in human trophoblast - a review. II. extravillous trophoblast.

At the tips of anchoring villi, cytotrophoblast (CTB) proliferation leads to a process of multilayering in which cells lose their attachment to the villous basement membrane and develop into columns, within which they adhere to one another using desmosomes, with associated intermediate filament bundles. Non-desmosomal cadherins, tight junction proteins and other adhesion molecules are also present, suggesting that actin-associated adhesions contribute to placental anchorage. In the distal columns, cell-cell interactions diminish, cells upregulate beta1 integrins and bind to a provisional fibrinoid extracellular matrix, eventually detaching to migrate into the decidual stroma and myometrium, where interstitial and endovascular extravillous trophoblast (EVT) populations show distinct repertoires of adhesion molecules.

Adhesion molecules in human trophoblast - a review. I. Villous trophoblast.

In the placental villus, cells attach to basement membrane via integrin alpha6beta4 and adhere both laterally and apically to their neighbours. The most prominent adhesive specialisation seen using the electron microscope is the desmosome, which connects cytotrophoblast cells (CTB) laterally and also contributes to the attachment of CTB to the overlying syncytium. However, numerous cadherins and other junctional proteins are also present in the corresponding plasma membrane domains, indicating a multiplicity of adhesive interactions. Integrins, tight junction components and cadherins are all found in the syncytial microvillous membrane, perhaps reflecting its ability to form intersyncytial bridges. There is a wide gulf to be filled between molecular anatomy and functional studies, with much to be learned about the role of adhesion molecules in regulating villous epithelial integrity, homeostasis and growth.

Methods for siRNA-mediated reduction of mRNA and protein expression in human placental explants, isolated primary cells and cell lines.

The use of RNA interference (RNAi) to deplete individual proteins from cells or tissue has revolutionised our ability to characterise gene function. The placenta is an attractive target for studies in which the role of specific proteins can be compared with cell culture models and explanted villous tissue where physiological function can be maintained ex vivo. In this study, we compared a variety of commercially available reagents and approaches to define methods for efficient delivery of siRNA to placental cells. Protocols optimised using fluorescently-labelled siRNA were subsequently tested using siRNA sequences that target placental alkaline phosphatase (PLAP), chosen because of its high abundance in trophoblast. mRNA abundance was assayed using qRT-PCR, and the effect on protein was examined using immunolocalisation. We report that different protocols are required for BeWo choriocarcinoma cells (nucleofection), primary cytotrophoblast cells (lipid-based transfection) and villous tissue explants (nucleofection). The results provide guidelines for optimal siRNA-mediated knockdown in these three models of the human placenta.

Integrin alpha 6/beta 4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion.

The alpha 6/beta 4 complex is a member of the integrin family of adhesion receptors. It is found on a variety of epithelial cell types, but is most strongly expressed on stratified squamous epithelia. Fluorescent antibody staining of human epidermis suggests that the beta 4 subunit is strongly localized to the basal region showing a similar distribution to that of the 230-kD bullous pemphigoid antigen. The alpha 6 subunit is also strongly localized to the basal region but in addition is present over the entire surfaces of basal cells and some cells in the immediate suprabasal region. By contrast staining for beta 1, alpha 2, and alpha 3 subunits was very weak basally, but strong on all other surfaces of basal epidermal cells. These results suggest that different integrin complexes play differing roles in cell-cell and cell-matrix adhesion in the epidermis. Immunoelectron microscopy showed that the alpha 6/beta 4 complex at the basal epidermal surface is strongly localized to hemidesmosomes. This result provides the first well-characterized monoclonal antibody markers for hemidesmosomes and suggests that the alpha 6/beta 4 complex plays a major role in epidermal cell-basement membrane adhesion. We suggest that the cytoplasmic domains of these transmembrane glycoproteins may contribute to the structure of hemidesmosomal plaques. Immunoultrastructural localization of the BP antigen suggests that it may be involved in bridging between hemidesmosomal plaques and keratin intermediate filaments of the cytoskeleton.

Trophoblast-derived heparanase is not required for invasion.

To invade the decidua and myometrium, extravillous trophoblast must degrade an assortment of extracellular matrix (ECM) components. The uterine wall is rich in heparan sulphate proteoglycans (HSPG), which interact with collagen, laminin and fibronectin, and bind a variety of growth factors. HSPG are catabolised by heparanase, an enzyme that is highly expressed in the placenta. The aim of this study was to investigate the role of heparanase in first trimester trophoblast invasion. First trimester cytotrophoblasts (CTB) were isolated by trypsin digestion followed by centrifugation on a Percoll gradient. Cells were cultured on Matrigel to promote an extravillous phenotype. Heparanase expression was studied by immunohistochemistry and confocal microscopy. Trophoblast invasion was assessed using an in vitro transwell assay. A high level of heparanase was observed in isolated first trimester trophoblast; however, a function-blocking antibody did not inhibit invasion of primary CTB or the extravillous trophoblast cell line SGHPL-4 at 21% oxygen. In contrast to cancer cells, heparanase expression was not increased following culture at 3% oxygen, and trophoblast invasion was not retarded by the blocking antibody under these conditions. Heparanase expression was observed in stromal cells and vascular endothelium in first trimester parietal decidua. Expression was evident on the cell surface and in the nucleus of trophoblast and decidual cells. In conclusion, trophoblast heparanase is not required for invasion in vitro. Its abundant expression suggests another role during pregnancy, perhaps in controlling the availability of ECM-bound growth factors or acting as a transcription factor.

A re-appraisal of the morphophenotype and basal lamina coverage of cytotrophoblasts in human term placenta.

A recent study of human placental villi [Mori et al., The cytotrophoblast layer of human chorionic villi becomes thinner but maintains its structural integrity during gestation, Biol Reprod 76 (2007) 164-172] concluded that cytotrophoblast (CT) cells occupy 80% of the basal lamina (BL) surface at term and that syncytiotrophoblast (ST) does not make direct contact with the BL. Based on SPINT-1 localisation using immunofluorescence on cryosections, these conclusions run counter to previous light and electron microscopic data suggesting that term CT cells cover no more than about 24% of the BL surface. To resolve these discrepancies, we have undertaken a stereological study of term placenta using transmission electron microscopy (TEM) and a novel immunofluorescence approach. Test line lattices were randomly superimposed on TEM images of villous trophoblast from 13 normal term placentae. Intersections with the test lines were counted to assess the fractional surface of BL occupied by CT cells. After trypsin-mediated removal of syncytium, cells in whole-mounted term and first trimester villi were stained with cytokeratin 7 to identify CT and then visualised by confocal microscopy. CT formed an almost continuous layer in the first trimester. In contrast, term CT cells and their processes were found to cover only 44% (SD 14%) of the BL surface with intervening regions occupied by ST. TEM and confocal images were consistent with the concept of a network of 'octopoid' CT cells with fine processes extending from a central cell body. Our estimates of CT coverage are lower than the recent immunofluorescence estimate but greater than earlier TEM estimates. The former may have been biased by overprojection (section thickness) effects whilst the latter may be underestimates due to failure to include the fine CT cell processes. We conclude that CT cells transform from a cuboidal phenotype early in gestation to flattened cells with multiple interconnecting processes. The CT layer thins but maintains a functional network within which cells intercommunicate without compromising substance transfer via the syncytium.

Embryo implantation: the molecular mechanism remains elusive.

Low rates of implantation are an impediment to more efficient assisted reproduction techniques. Improved endometrial receptivity and embryo preparation should lead to higher pregnancy rates, lower rates of early pregnancy failure and fewer multiple pregnancies. As the first site of contact between embryo and endometrium, the luminal epithelium (LE) is responsible for the non-receptive status of proliferative and early secretory tissue, and transformation to receptivity in the mid-secretory phase presumably requires alterations in expression, organization or activation of adhesion systems. Luminal cells are less abundant than their glandular counterparts, and are under-represented in global tissue datasets. Furthermore, alterations in cell surface composition can be readily accomplished by mechanisms that do not rely on altered transcription or translation. Current data from in-vitro models are consistent with initial attachment to mucin in the apical glycocalyx, perhaps via a carbohydrate-mediated interaction, after which the epithelial phenotype is modified by a medium- or short-range embryonic signal. A cascade of interactions follows, mediating embryo migration across the epithelium. Strikingly, numerous potential mediators of adhesion at implantation are located in the lateral rather than the apical surface of LE cells. Attached embryos appear to gain rapid access to this highly adhesive lateral membrane domain.

Trophoblast differentiation: progenitor cells, fusion and migration -- a workshop report.

Challenge lies ahead in unravelling the role played by trophoblast and its repertoire of expressed genes in normal human placental development, growth and pathology. Specific technical advances will clearly be required for characterisation of function. In particular, improvements in our repertoire of in vitro models are needed before many of the key questions can be answered. Recent advances in the study of human trophoblast differentiation are discussed.

Human placental explants in culture: approaches and assessments.

Placental explant cultures in vitro are useful for studying tissue functions including cellular uptake, production and release of secretory components, cell interactions, proliferation, growth and differentiation, gene delivery, pharmacology, toxicology, and disease processes. A variety of culture conditions are required to mimic in utero environments at different times of gestation including differing oxygen partial pressures, extracellular matrices and culture medium. Optimization of explant methods is examined for first and third trimester human placental tissue and the biological processes under investigation.

Tracking nutrient transfer at the human maternofetal interface from 4 weeks to term.

INTRODUCTION: In this study we have tracked glycogen and glycoprotein flux associated with nutrient uptake into trophoblast in early deciduochorial and later haemochorial placenta. METHODS: α-amylase, glycogen synthase and glycogen phosphorylase were immunohistochemically localised in 6-14 week and term placenta and first trimester decidua. Placentae of 4-18 weeks' gestation and term were also stained with 22 biotinylated lectins. RESULTS: Histochemical data were consistent with glycogenolysis in decidual gland epithelium and placental cyto- and syncytiotrophoblast; α-amylase was present in decidual secretions but absent in placenta. Glycogen and glycogen synthase were both apparent in villous cytotrophoblast cells and columns. Profound changes were observed in placental glycosylation during gestation. Syncytial microvilli were richly glycosylated as were first trimester vacuoles but, by term, syncytiotrophoblast showed little lectin binding except in microvillous and basal membranes. Cytotrophoblast Golgi bodies were active in the first trimester; at term the cells were generally more glycosylated than syncytiotrophoblast. DISCUSSION: We deduce that decidual cell glycogen is broken down for transport into the placenta where the products may be reassembled into glycogen or used for metabolic processes. First trimester histiotrophe is internalised by syncytiotrophoblast, then broken down in apical vacuoles containing lysosomal markers. This process declines after haemotrophic nutrition commences. Transition from histiotrophic to haemotrophic nutrition involves reduced amounts of uterine secretory derivatives reaching the placenta, and reduction in internalisation of glycoprotein by syncytiotrophoblast, presumably reflecting the shift to low molecular weight nutrients. Glycogen accumulates in cytotrophoblast from early pregnancy and is mobilised for utilisation by fetoplacental tissues.

Novel substratum-dependent dendritic morphology and motility in epithelial cells.

Qualitative and quantitative light microscopy including time-lapse observations, and scanning electron microscopy are applied to study the response of cultured primary amnion epithelial cells to surfaces coated with a fibronectin-containing aggregate extracted from placenta (PSF). Cell shapes are compared with those observed in medium containing serum and amniotic fluid. Cells respond rapidly to PSF in serum-free medium to give unusual dendritic structures. Cell bodies remain rounded with numerous long and complex processes appearing and disappearing over a period of 6 to 6 1/2 h. This behaviour is associated with an 18-fold increased motility relative to control cells. Few attachments to substratum occur beneath cell bodies or along dendritic processes, points of adhesion being largely confined to the termini of extensions. Later in the response to PSF, a reduction in cell extensions is accompanied by flattening of the cell body and more conventional shapes reminiscent of control cells are restored. In serum, cells spread more slowly to give well-flattened discoid or polygonal morphologies. In amniotic fluid, blebbed, bipolar and asymmetrical cells are observed with some dendritic extensions, reminiscent of those seen on PSF. The profound influence of substratum-associated ligands on cellular behavior is noted.

Cell surface molecules involved in fibronectin-mediated adhesion. A study using specific antisera.

Attachment and spreading of baby hamster kidney (BHK) fibroblasts to fibronectin-coated surfaces can be inhibited by antibodies, and the (Fab) 2 fragments, prepared against the cells. The antibodies reacted specifically with ricin-binding glycoproteins of the cell surface, the major components having molecular weights of 130-140 K. The antibodies reacted also with mouse fibroblasts (L-cells) and Chinese hamster ovary (CHO)-cells and inhibited their fibronectin-mediated adhesion to inert surfaces. Antisera raised against material isolated from the underside of BHK cells spread out on fibronectin-coated substrata using a cleavable, photolabile heterobifunctional cross-linking reagent (Aplin et al. Exptl. Cell Res. (1981) 134, 488-494) also affected the interactions of cells with fibronectin-coated surfaces. The antibodies stained the periphery of BHK cells and L-cells in a discontinuous periodic manner and immunoprecipitated as a major component a low molecular weight (approximately 47 K) glycoprotein from cell extracts. These results indicate that different specific antisera modify cell-substratum adhesion, probably by interacting with different cell surface components. The properties of multiple factors involved in the attachment and spreading of cells to suitably adhesive substrata are discussed.

Comment: effect of cytokines on prolactin production by human decidual stromal cells in culture: studies using cells freed of bone marrow-derived contaminants.

Human decidua contains resident decidual cells alongside a population of bone marrow-derived cells, among which macrophages and large granular lymphocytes are most abundant. We hypothesized that soluble effectors produced by bone marrow-derived cells may modulate the function of the decidual cells. To investigate this, a cell purification protocol was devised that involved digestion of first-trimester decidua with collagenase and hyaluronidase to produce a mixed stromal cell suspension from which the bone marrow-derived cells were removed using immunomagnetic beads coated with anti-CD45. The resulting stromal cells were maintained in culture in the presence of progesterone and were found to produce PRL. The effect of a panel of cytokines on PRL production was examined. Tumor necrosis factors-alpha and -beta had a dose-dependent inhibitory effect, and tumor necrosis factor receptors were identified on the cells. Interleukin 1 alpha and 1 beta, platelet-derived growth factor, and transforming growth factor-beta 1 were also found to inhibit PRL production, and platelet-derived growth factor and transforming growth factor-beta 1 stimulated cell proliferation. These findings suggest an interaction between the immune and endocrine systems in regulating the maternal environment of early pregnancy.